Structural Variability of the Initiation Complex of HIV-1 Reverse Transcription

Abstract

HIV-1 reverse transcription is initiated from a tRNA 3Lys molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA 3Lys are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA 3 Lys annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA 3Lys are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA 3 Lys annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA His ). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

“…In the PBS domain, we observed the interaction of tRNA 3 Lys with the PBS but none of the proposed additional interactions (19,20,39,66). This result is in keeping with a recent comparative in vitro and ex vivo comparison of the HIV-1 NL4.3 and MAL isolates (41), which suggested that no interaction between the genomic RNA and tRNA 3 Lys is required outside the PBS for optimal initiation of reverse transcription of subtype B isolates. Finally, our results do not favor the proposed LDI conformer (6,8,9,11), and although one cannot exclude that minor conformations are present at low concentration or with a short half-life, we managed to test the major conformation in intact cells and virions without the need for RNA renaturation and avoiding any possible artifacts because of the ionic conditions or the short length of RNA transcripts.…”

“…Taken together, these data indicated that HXB2NEO genomic RNA underwent little, if any, structural rearrangement during formation of the reverse transcription initiation complex. This conclusion is in keeping with comparative in vitro and ex vivo structural probing and in vitro functional comparison of the HIV-1 NL4.3 and MAL isolates (41). This study suggests that the interaction between the A-loop and the anticodon loop of tRNA 3 Lys only takes place in a subset of HIV-1 isolates, including MAL, whereas no interaction between the viral RNA and the primer tRNA is required outside the PBS for optimal initiation of reverse transcription of subtype B isolates, including NL4.3 and HXB2.…”

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

“…7). As the sequence and structure of the upper part of the PBS domain significantly differ between the HIV-1 NL4.3 and MAL isolates 49 , it is possible that either this region of the RNA is not involved in negative regulation or these two isolates use different mechanisms to negatively regulate Pr55 Gag binding. We suggest that the negative effect is due to steric hindrance that prevents binding of Pr55 Gag to the lower part of SL1 (Fig.…”

“…(For clarity, all RNAs derived from NL4-3 have a name starting with 'N', while those derived from MAL have a name starting with 'M'). The reason for using MAL RNAs is twofold: first, several mutants of this RNA were already available in our laboratory; second, this isolate possesses an insertion 3 0 to the PBS that is frequent in the HIV-1 circulating recombinant forms and in subgroup G and A isolates 49 (as a consequence, the SD1 site of the MAL gRNA is located between positions 305 and 306). Importantly, the structural differences in the 5 0 -region of the gRNA of these two isolates reside in the upper part of the PBS domain 49 .…”

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

“…2). Structure-probing studies of HXB2 (a clade B isolate) and MAL (a mosaic of clades A, D, and I) concluded that the two viruses have different PBS stem folds (Goldschmidt et al 2004). In HXB2, the U-rich anti-codonlike element is an exposed 7-nt loop (Fig.…”